The goal of this project is to determine the complete sequence of the chromosome of Staphylococcus aureus, one of the leading causes of both hospital and community-acquired infections worldwide. Infections caused by S. aureus are often acute and, in the case of highly invasive strains and/or an immunocompromised host, S. aureus is able to overcome the local phagocytic systems and invade the bloodstream where it can subsequently colonize any tissue and become life-threatening. With the emergence of methicillin resistant S. aureus (MRSA) in the 1980's, vancomycin became the last uniformly effective antibiotic for serious S. aureus infections. However, two documented cases of vancomycin resistance in 1997 may signal the emergence of S. a aureus strains with compete resistance to vancomycin. Vancomycin resistance, coupled with the lack of effective vaccine candidates, represents one of the most frightening challenges in antibiotic resistance. Clearly, the complete genomic sequence and a set of recombinant clones would provide a tremendous resource for the study of S. aureus. Recent advances in large scale genome sequencing, assembly, and analysis have made this a feasible, cost-effective approach towards this goal. The approach will be the whole-genome random-sequencing strategy successfully used at TIGR to completely sequence seven prokaryotic genomes. The project will consist of three phases; (1) construction of a random small insert plasmid library from a recent clinical isolate of S. aureus, (2) sequencing both ends of approximately 24,000 clones (48,000 sequence fragments), (3) sequencing the ends of a set of minimally overlapping lambda clones to provide a scaffolding structure that will minimize the effort required for gap closure and provide confirmation of the underlying assembled structure, and (4) assembly and annotation of the genome to identify structural features, assign gene and functional roles to open reading frames based on database similarity searches. This approach will impact the development of vaccines and expedite the discovery of new targets for antimicrobial therapy. The discovery of novel virulence factors and their global regulatory networks will enhance our understanding of the biology and pathogenic mechanisms of S. aureus. The data developed from this study will be deposited into a variety of databases including the TIGR World Wide site. In addition, small-insert plasmid and lambda clone sets will be made available to the research community.